Methods and compositions for obtaining marker-free transgenic plants

ABSTRACT

The invention provides methods and compositions for identifying transgenic seed that contain a transgene of interest, but lack a marker gene. Use of an identification sequence that results in a detectable phenotype increases the efficiency of screening for seed and plants in which transgene sequences not linked to a gene of interest have segregated from the sequence encoding a gene of interest.

This application claims the priority of U.S. Provisional ApplicationSer. No. 60/799,875, filed May 12, 2006, the entire disclosure of whichis incorporated herein by reference.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The invention generally relates to transgenic plants. More specifically,the invention relates to identification and removal of unwanted orunnecessary DNA in transformed plants.

2. Description of Related Art

The identification of unnecessary or unwanted transgenic DNA intransformed plants has been the subject of numerous investigations andmany different methods have been examined in efforts to eliminate thesetransgenic sequences from such plants (e.g. Hanson et al., 1999; Dale etal., 1991; Ebinuma et al., 1997; Yoder et al., 1994; Kononov et. al.,1997; Hare and Chua, 2002; Scutt et al., 2002; Puchta, 2003; de Vettenet al., 2003; Halpin, 2005; U.S. Published Appln. 20030110532; U.S.Published Appln. 20040237142; U.S. Pat. No. 6,458,594). In general, itis beneficial to identify plants that do not include transgenic DNA notcontributing to an agronomically useful trait of the transgenic plant.

Many methods for introducing transgenes in plants byAgrobacterium-mediated transformation utilize a T-DNA (transferred DNA)that incorporates a transgene and associated genetic elements, andtransfers these into the genome of a plant. Generally, the transgene(s)is bordered by a right border DNA molecule (RB) and a left border DNAmolecule (LB), and is transferred into the plant genome, integrating atone or more loci. It has been observed that when a DNA constructcontains more than one T-DNA, these T-DNAs and the transgenes containedwithin may be integrated into the plant genome at separate loci (Framondet al., 1986). This is referred to as co-transformation.

The process of co-transformation can be achieved by delivery of theT-DNAs with a mixture of Agrobacterium strains transformed with plasmidscarrying the separate T-DNAs. Co-transformation can also be achieved bytransforming one Agrobacterium strain with two or more DNA constructs,each containing one T-DNA. An additional method employs two T-DNAs on asingle DNA vector and identifying transgenic cells or plants that haveintegrated the T-DNAs at different loci. In a non-Agrobacterium-mediatedtransformation system, such as a physical method for introducing DNAincluding bombardment with microprojectiles, two DNA molecules could beintegrated independently into the target genome, and then segregateindependently in a subsequent generation. Use of 2 T-DNA constructsallowing for independent insertion of sequences and their geneticsegregation, has also been described (e.g. U.S. Pat. No. 5,731,179; Zhouet al., 2003; Breitler et al., 2004; Sato et al., 2004). While theforegoing has furthered the understanding in the art, there remains aneed for improved methods and compositions for obtaining marker freeplants to make product development more efficient. Previously describedscreening processes have been highly labor intensive, for instancerequiring Southern blot or PCR™ analysis following growth of R0 and/orR1 plant material.

U.S. Publication 20060041956 describes use of a visual marker gene inconjunction with Agrobacterium-mediated transformation. However, thepublication does not describe any method where such markers are linkedto a selectable or screenable marker gene and unlinked to a gene ofinterest. Thus, there remains a great need in the art for methods andcompositions that would improve the ease and efficiency with whichplants lacking marker sequences and/or other transgenic DNA which is notagronomically useful can be identified and eliminated.

SUMMARY OF THE INVENTION

In one aspect, the invention provides a method of preparing marker-freeseeds from a transgenic plant comprising the steps of: a) obtainingseeds of a transgenic plant transformed with a first DNA segmentcomprising a nucleic acid of interest and a second DNA segmentcomprising a plant marker gene physically and/or genetically linked to aDNA cassette that is operably linked to a promoter functional in theseed, wherein the DNA cassette confers a detectable phenotype to seedsthat comprise the DNA cassette; b) screening the seeds for the absenceof the detectable phenotype; and c) selecting at least a first seed thatlacks the detectable phenotype to obtain a seed that is free of themarker gene. In one embodiment, step c) further comprises assaying theseed for the presence of the nucleic acid of interest and selecting aseed that comprises the nucleic acid of interest and lacks theselectable marker gene. In certain embodiments, the marker gene is aselectable or screenable marker gene.

In certain embodiments, the DNA cassette may be translationally ortranscriptionally fused to the selectable marker gene; that is, it mayencode an RNA that is translationally or transcriptionally fused to theselectable marker gene. In a further embodiment the DNA cassettecomprises an antisense or sense DNA fragment with at least 19 or 21 bpof homology to an endogenous gene, for instance wherein the antisense orsense DNA fragment is operably linked to a promoter functional in aseed. In yet another embodiment, the DNA cassette comprises a pair ofinverted repeats of a DNA fragment, wherein each fragment is at least 19or 21 bp in size, and wherein the DNA fragment is homologous to anendogenous gene, operably linked to a promoter functional in the seed.The inverted DNA fragment repeat homologous to an endogenous gene mayalso be embedded in an intron within the selectable marker gene. Incertain embodiments, the DNA cassette encodes a sense or antisense RNAcomprising at least 19 or 21 nucleotides wherein the DNA fragment ishomologous to an endogenous gene.

In a method of preparing marker-free seeds according to the invention,seed selected may lack a screenable or screenable gene and DNA cassette.Obtaining seeds of a transgenic plant may comprise transforming orco-transforming the transgenic plant or a progenitor thereof of anyprevious generation with first and second DNA segments on separate DNAconstructs. Obtaining seeds of a transgenic plant may also comprisetransforming the transgenic plant or a progenitor thereof of anyprevious generation with a single DNA construct comprising the first andsecond DNA segments. First and second DNA segments may be bounded bydifferent T-DNA border sequences. In a method of the invention, atransgenic plant may be produced by transforming the plant or aprogenitor thereof of any previous generation with a DNA constructcomprising (i) the first DNA segment flanked by left and right T-DNAborders, and (ii) the second DNA segment flanked by a second set of leftand right T-DNA borders, wherein the second DNA segment furthercomprises a selectable marker gene operably linked to a promoterfunctional in the transgenic plant. The first and second DNA segmentsmay or may not be genetically linked in the transgenic plant.

Transgenic plants used according to the invention may be produced byintroducing first and second DNA segments into the plant or a progenitorthereof of any previous generation by transformation mediated by abacterial strain selected from the genus Agrobacterium, Rhizobium,Mesorhizobium, or Sinorhizobium. The transgenic plants may also beproduced, for example, by microprojectile bombardment.

A selectable marker used with the invention may encode a productselected from the group consisting of CP4 EPSPS, bar, DMO, NptII,glyphosate acetyl transferase, mutant acetolactate synthase,methotrexate resistant DHFR, dalapon dehalogenase, PMI, Protox,hygromycin phosphotransferase and 5-methyl tryptophan resistantanthranilate synthase. A DNA cassette sequence for use with theinvention may be selected, for example, from the group consisting ofcrtB, gus, gfp, sacB, lux, an anthocyanin synthesis gene, DefH9-iaaM,rolB, OsCDPK2, AP2, AFR2, ANT transcription factor, LEC2, Snf-1, cobA,KAS4, splA, zein inverted repeats, B-peru, and yeast ATP-PFK. Thecassette may be operably linked to a promoter functional in a tissueselected from an embryo, seed endosperm, cotyledon, aleurone, and seedcoat. The promoter may be, for example, selected from the groupconsisting of a napin promoter, a beta-phaseolin promoter, abeta-conglycinin subunit promoter, a zein promoter, an Osgt-1 promoter,an oleosin promoter, a starch synthase promoter, a globulin 1 promoter,a barley LTP2 promoter, an alpha-amylase promoter, a chitinase promoter,a beta-glucanase promoter, a cysteine proteinase promoter, aglutaredoxin promoter, a HVA1 promoter, a serine carboxypeptidase IIpromoter, a catalase promoter, an alpha-glucosidase promoter, abeta-amylase promoter, a VP1 promoter, a USP promoter, USP88 promoter,USP99 promoter, Lectin, and a bronze2 promoter. The detectable phenotypemay be assayed by detection of a catalytic activity. The detectablephenotype may be selected from the group consisting of seed color, seedopacity, seed germinability, seed size, seed viability, seed shape, seedtexture, and a defective or aborted seed. Screening of seeds may be doneby an automated seed sorting machine.

In another aspect, the invention provides a DNA construct comprising (a)a first DNA segment comprising left and right T-DNA borders flanking agene of interest operably linked to a promoter functional in plants, and(b) a second DNA segment comprising a second set of left and right T-DNAborders flanking a promoter functional in a seed operably linked to aDNA cassette that confers a detectable phenotype in seeds comprising theDNA cassette and a selectable marker gene operably linked to a promoterfunctional in plants. The gene of interest may confer a trait selectedfrom the group consisting of herbicide tolerance, insect or pestresistance, disease resistance, increased biomass, modified fatty acidmetabolism, modified carbohydrate metabolism, and modified nutritionalquality. In the construct, the DNA cassette and selectable marker genemay be operably linked to the same promoter. In one embodiment, the DNAcassette and the selectable marker gene are operably linked to differentpromoters. In specific embodiments, the selectable marker gene encodes aproduct selected from the group consisting of CP4 EPSPS,phosphinothricin acetyltransferase, DMO, NptII, glyphosate acetyltransferase, mutant acetolactate synthase, methotrexate resistant DHFR,dalapon dehalogenase, PMI, Protox, hygromycin phosphotransferase and5-methyl tryptophan resistant anthranilate synthase. In anotherembodiments, the DNA cassette is selected from the group consisting ofcrtB, gus, gfp, sacB, lux, an anthocyanin synthesis gene, DefH9-iaaM,rolB, OsCDPK2, AP2, AFR2, ANT transcription factor, LEC2, Snf-1, cobA,KAS4, splA, zein inverted repeats, B-peru, and yeast ATP-PFK. The DNAcassette may be operably linked to a promoter functional in a tissueselected from the group consisting of an embryo, seed endosperm,cotyledon, aleurone, and seed coat. In one embodiment, the DNA cassetteis operably linked to a promoter selected from the group consisting of anapin promoter, a beta-phaseolin promoter, a beta-conglycinin subunitpromoter, a zein promoter, an Osgt-1 promoter, an oleosin promoter, astarch synthase promoter, a globulin 1 promoter, a barley LTP2 promoter,an alpha-amylase promoter, a chitinase promoter, a beta-glucanasepromoter, a cysteine proteinase promoter, a glutaredoxin promoter, aHVA1 promoter, a serine carboxypeptidase II promoter, a catalasepromoter, an alpha-glucosidase promoter, a beta-amylase promoter, a VP1promoter, a USP88 or USP99 promoter, and a bronze2 promoter.

In yet another aspect, the invention provides transgenic cells andplants transformed with a construct provided herein. In one embodiment,a transgenic plant is provided that is co-transformed with a DNAconstruct containing a first DNA segment comprising left and right T-DNAborders flanking a gene of interest operably linked to a promoterfunctional in plants and a second DNA construct containing a second DNAsegment comprising a second set of left and right T-DNA borders flankinga promoter functional in a seed operably linked to a DNA cassette thatconfers a detectable phenotype in seeds comprising the DNA cassette anda selectable marker gene operably linked to a promoter functional inplants. Cells of such a plant are also provided.

In still yet another aspect, the invention provides a DNA constructcomprising right and left T-DNA borders, wherein a first DNA segmentcomprising a gene of interest operably linked to a promoter functionalin plants is located after the right border and a second DNA segmentcomprising a DNA cassette that confers a detectable phenotype to plantseeds that comprise the DNA cassette and a marker gene, such as aselectable marker gene, operably linked to a promoter functional inplants is located after the left border.

In still yet another aspect, the invention provides a DNA constructcomprising right and left T-DNA borders, wherein a first DNA segmentcomprising a DNA cassette that confers a detectable phenotype to plantseeds that comprise the DNA cassette and a selectable marker geneoperably linked to a promoter functional in plants is located after theright border and a second DNA segment comprising a gene of interestoperably linked to a promoter functional in plants is located after theleft border.

In still yet another aspect, the invention provides a DNA constructcontaining two right T-DNA borders, wherein a first DNA segmentcomprising a gene of interest operably linked to a promoter functionalin plants is located after one right border and a second DNA segmentcomprising a DNA cassette that confers a detectable phenotype to plantseeds that comprise the DNA cassette and a selectable marker geneoperably linked to a promoter functional in plants located after theother right border.

In yet another aspect, the invention provides an isolated nucleic acidsequence comprising SEQ ID NO:2, SEQ ID NO:3, or a sequence with atleast 70%, 75%, 85%, or 95% identity to SEQ ID NO:2 or SEQ ID NO:3, andencoding a polypeptide with phytoene synthase activity. In oneembodiment, the invention also provides a recombinant DNA constructcomprising a nucleic acid sequence of SEQ ID NO:2 or SEQ ID NO:3, or arecombinant DNA construct comprising a sequence with at least 71%, 80%,90%, 95%, 98%, or 99% identity to SEQ ID NO:2 or SEQ ID NO:3, andencoding a polypeptide with phytoene synthase activity, operably linkedto a heterologous promoter functional in a plant. A host cell comprisingsuch a sequence, wherein the cell is a bacterial cell or a plant cell isanother embodiment of the invention. In another embodiment, theinvention provides a transgenic plant or seed comprising SEQ ID NO:2 orSEQ ID NO:3 SEQ ID NO:2, SEQ ID NO:3, or a sequence with at least 71%,80%, 90%, 95%, 98%, or 99% identity to SEQ ID NO:2 or SEQ ID NO:3, andencoding a polypeptide with phytoene synthase activity.

BRIEF DESCRIPTION OF THE DRAWINGS

The following drawings are part of the present specification and areincluded to further demonstrate certain aspects of the presentinvention. The invention may be better understood by reference to thedrawings in combination with the detailed description of specificembodiments presented herein.

FIG. 1. Schematic diagrams of: (A) pMON10338; (B) pMON10339; and (C)pMON67465.

FIG. 2. CrtB expression in soybean tissues transformed with pMON67465.

FIG. 3. CrtB expression in seed from event A33908.

FIG. 4. Expression of crtB, gus, and CP4/EPSPS in immature R1 seed.

FIG. 5. Expression of crtB in mature R1 seed.

FIG. 6. GUS staining of pMON67465 seed.

FIG. 7. CP4 & CrtB PCR on GUS positive seeds.

FIG. 8. Comparison of linkage-Southern and screenable-marker approachesfor screening transgenic events.

FIG. 9. Schematic summary of DNA sequences transferred by use ofconstruct comprising a screenable gene linked to CP4 selectable markergenes for marker-free seeds. A) GOI located in one T-DNA flanked with aRB and LB and physically linked to a second T-DNA containing ascreenable gene linked to a CP4 selectable marker gene in one constructused for Agrobacterium-mediated transformation; B) One vector containingtwo borders, the GOI is placed after a RB while the screenable andselectable marker genes are placed after the second RB or after a LBtogether with backbone; C) The GOI and screenable genes—DNAs areseparated in two vectors and transformed in either one Agrobacteriumcell or separate Agrobacterium cells; D) Possible linkage of two DNAsegments from the GOI and screenable and selectable marker genes. Onlythe GOI alone will show normal seed appearance, while cells containingthe screenable gene show a visible phenotype; E) Two separate DNAsegments contain either the GOI or screenable and selectable markergenes used for non-bacterial mediated transformation.

FIG. 10. pMON67420 represents a GOI construct for co-transformation.

FIG. 11. Plasmid pMON99575 containing Schizosaccharomyces pombe ATPdependent phosphofructokinase driven by the seed-specific zein promoter.

FIG. 12. Corn ear expressing seed-specific yeast ATP dependentphosphofructokinase abolished normal kernel development.

FIG. 13. Schematic diagram of dsRNA-encoding constructs used todemonstrate that inverted repeats placed within an intron of a markergene result in a visible phenotype.

FIG. 14. Inverted repeats embedded in an intron give rise to a visiblephenotype.

FIG. 15. Silencing of α-zeins in corn kernels leads to a visiblephenotype.

FIG. 16. Schematic diagram of pMON83530 containing KAS4.

FIG. 17. Progeny of soybean seeds transformed with pMON83530. Seeds onthe left are shrunken due to expression of KAS4 and indicate thepresence of selectable marker, while seeds on the right are normal andmarker-free.

FIG. 18. Schematic diagram of pMON107314 containing KAS4 useful as anidentification sequence in a 2T DNA construct.

FIG. 19. Schematic diagram of pMON68581 containing a splA gene useful asan identification gene.

FIG. 20. Progeny soybean seeds transformed with pMON68581. Seeds on theright are shrunken due to the expression of splA and indicate thepresence of the screenable or selectable marker, while seeds on the leftare normal and marker-free.

FIG. 21. 2 T-DNA vector formats-schematic diagram.

DETAILED DESCRIPTION OF THE INVENTION

The following is a detailed description of the invention provided to aidthose skilled in the art in practicing the present invention. Those ofordinary skill in the art may make modifications and variations in theembodiments described herein without departing from the spirit or scopeof the present invention.

The invention overcomes deficiencies in the prior art by providingconstructs and methods allowing for efficiently distinguishing seedslacking an identification sequence and marker-gene sequence, butincluding a gene or genes of interest (GOI), from seeds that contain anidentification gene sequence and marker-gene sequence, based on aphenotype conferred by a seed-expressed identification gene, sequence,or cassette. In particular, the present invention provides, in oneembodiment, transformation constructs and methods for transformation ofplant cells which include: (i) a gene of interest; and (ii) anidentification sequence expressed in seed and physically linked to aselectable or screenable marker gene that may be expressed in variousplant tissues, wherein the construct and/or transformation method isdesigned so that the genetic elements of (i) and (ii) can integrateindependently into the plant genome, and thus genetically segregate fromeach other.

The expression or lack thereof of the identification sequence in seedtissues allows for direct identification of seeds and plants that lackthe seed-expressed identification sequence and the physically linkedselectable or screenable marker gene, while allowing for choice of seedand plants still containing the transgene of interest. Choosing seedincluding the gene of interest and lacking marker-gene sequences at theseed level represents a significant advance in that it avoids the needfor previously utilized screening methods that are comparatively costand labor intensive. Additionally, time may be saved as screening can bedone prior to or during germination without requiring growth of the nextgeneration of plants to a size permitting tissue harvest, as needed forlinkage-Southern analysis, for example.

In one embodiment, transformation of plant tissue is performed by anAgrobacterium or other Rhizobia-mediated method (See e.g., U.S.Provisional Patent Application Ser. No. 60/800,872, filed May 16, 2006,entitled “Use of Non-Agrobacterium Bacterial Species for PlantTransformation” and assigned attorney docket No. MONS:100USP1, theentire disclosure of which is specifically incorporated herein byreference), and the DNA sequences including the identification sequenceexpressed in seed and the physically linked selectable or screenablemarker gene are present together on a T-DNA or other sequence (e.g.,vector backbone) that is transferred into a plant cell (e.g. flanked byT-DNA RB and/or LB sequences or without a border sequence). Theidentification sequence and marker gene may be transferred to the plantphysically linked, while the gene of interest is present on a separateT-DNA flanked by its own RB and LB sequences, or other sequencetransferred into a plant cell, and can be integrated at an independentlocus (e.g. FIG. 21). The selectable and/or screenable marker permitsidentification of transformed plant tissues. Fertile plants can beobtained and selfed or crossed in a breeding scheme in order to followsegregation of phenotypes in the next generation. Strategies forperforming such breeding are well known in the art, and may vary indetails between different plants. Seed expression, or lack ofexpression, of the identification sequence permits facile identificationof seed with respect to the presence of marker and identificationsequences. The gene of interest, for example, may contain at least oneplant expression cassette encoding a trait selected from the groupconsisting of herbicide tolerance, antibiotic resistance, insectresistance, disease resistance, stress resistance (e.g, drought andcold), enhanced nutrient use efficiency, enhanced nutritive content(e.g., amino acid, protein, sugars, carbohydrates, fatty acids, and/oroil), sterility systems, industrial enzymes (e.g., pharmaceuticals andprocessing enzymes for bio-fuels) and enhanced yield.

The sequences that may be transferred into a plant cell (e.g. T-DNAs)may be present on one transformation vector in a bacterial strain beingutilized for transformation. In another embodiment, the sequencesincluding the identification sequence and plant selectable marker, andthe sequence(s) comprising the gene(s) of interest may be present onseparate transformation vectors in the bacterial strain. In yet anotherembodiment, the T-DNA including the identification sequence and plantselectable marker and the T-DNA comprising the gene of interest may befound in separate bacterial cells or strains used together fortransformation.

In still another embodiment, DNA sequences including the (i) gene ofinterest; and (ii) identification sequence expressed in seed andphysically linked to a selectable or screenable marker gene may beintroduced into a plant cell by a physical method such asmicroprojectile bombardment. In such an embodiment, the DNA sequences of(i) and (ii) can be located on separate DNA fragments that may be mixedtogether prior to or during the coating of microprojectiles with DNA.The DNA sequences may be present on a single microprojectile, or theymay be present on separate microprojectiles that are mixed togetherprior to bombardment.

The phenotype conveyed by the identification sequence can be achieved byectopic overexpression of a heterogenous or endogenous gene linked to aconstitutive or seed-specific promoter, or by downregulation of anendogenous gene using antisense RNA, RNA interference or co-suppressiontechnology. Examples of the endogenous gene may include, but are notlimited to, genes involved in sugar/starch metabolism, proteinmetabolism, and fatty acid metabolism.

In one embodiment, seed expression of the identification sequenceresults in a detectable phenotype in seed of a transgenic plantcontaining an identification sequence. In some embodiments the phenotypemay be detected by visual inspection, and may include a change in seedcolor, opacity (or translucence), fluorescence, texture, size, shape,germinability, viability, or generally any component or property that isphysically or biochemically assayable and different from that found inthe nontransgenic recipient genotype. In certain embodiments, theidentification sequence includes a gusA, gfp (Pang et al., 1996),phytoene synthase, or phytoene desaturase encoding gene, or ananthocyanin gene (Pl, Lc, B-Peru, C1, R, Re, mybA or mybl (e.g. Selingeret al., 1998; Ludwig et al., 1989; Himi et al., 2005; Kobayashi et al.,2002)). In a particular embodiment, the identification gene comprises acrtB gene encoding a phytoene synthase (U.S. Pat. Nos. 5,429,939; U.S.Pat. No. 6,429,356; U.S. Pat. No. 5,545,816), including a genecomprising a crtB sequence codon-optimized for expression in a monocotplant, such a corn plant. Another example of gene that could be used inthis regard is a gene involved in production of seed pigment.

In other embodiments, the phenotype is assayable by detection of acatalytic activity. In yet other embodiments, the phenotype is a tissueablation phenotype, for instance a blockage in the formation of pollen,egg, or seed tissue. Compositions and methods that silence genesrequired for the production or viability of gametes, reducing orpreventing fertilizations that include the marker gene, are alsoenvisioned. For example, sequences could be used that result in thesilencing of genes required for pollen development and viability. Thepollen that are derived from meiotic segregants carrying the marker genewould not develop or would be inviable, thus preventing the transmissionof the marker gene to the progeny through the pollen. In outcrosspollinations, all progeny would be marker free. Use of sequences thatresult in silencing of other endogenous genes (e.g. RNAi technologiesincluding miRNA) to result in a seed phenotype is also envisioned. Suchgenes include, but are not limited to: genes encoding or modifyingexpression of seed storage proteins such as zeins, Opaque2, Waxy, andother genes encoding proteins involved in carbohydrate, protein, and/orlipid accumulation in seeds.

Expression of an identification sequence that confers a phenotype ofnonviable pollen is also desirable because only the pollen grainswithout the identification sequence will be capable of fertilizing eggsthus increasing the yield of seeds free of the identification sequenceand the marker sequence when the transgenic line carrying theidentification sequence under the control of a pollen-specific promoteris used as a male pollinator. The identification sequence can produce aprotein that is lethal to the pollen or inhibitory to pollengermination. Alternatively, expression of a pair of inverted repeatshomologous to an essential endogenous pollen gene can be used to silencethe gene rendering the pollen nonviable. Examples of pollen specificgenes and promoters are known to those skilled in the art and includefor instance LAT52 and LAT59 genes and promoters of tomato as described(Eyal et al., 1995).

In another embodiment, the identification sequence can be expressed inboth the seed and the pollen for further enhancing the selection ofseeds with the gene of interest and eliminating the seeds with theidentification sequence and the marker gene. This can be achieved byusing an identification sequence comprised of two transgenes; the one ofwhich expresses in the seed and the other expresses in the pollen.Alternatively, a promoter that can express the same identificationsequence in the pollen and seed can also result in a detectablephenotype in both the pollen and the seed. Examples of promoters thatexpress in pollen and seed are the promoters from the maize Waxy gene(zmGBS; Shure et al., 1983), and the rice small subunit ADP-glucosepyrophosphorylase gene (osAGP; Anderson et al., 1991). Pollen and seedexpression patterns are also described in Russell and Fromm, 1997.

The identification sequence may alternatively alter carbohydrate,protein, lipid, or other products of cell or seed metabolism so as toyield a detectable phenotype. In one embodiment, the identificationsequence allows for endosperm-specific expression of a sacB geneencoding a levansucrase or a yeast ATP-dependent phosphofructokinase(ATP-PFK) which abolishes starch accumulation in seeds containing theidentification sequence and marker gene (Caimi et al., 1996; FIG. 12).The identification sequence can be a gene. The identification sequencecan further encode a transcriptional or translational fusion (e.g. U.S.Pat. No. 6,307,123; U.S. Patent Publication 20060064772).

U.S. Pat. No. 6,307,123 relates to the construction of a translationalfusion between a selectable marker gene (nptII) and a screenable markergene (gfp). The method can be applied to produce a fusion between anidentification sequence described herein and a selectable or ascreenable marker described herein.

Another method that can be used to make a polypeptide fusion is based onthe Ubiquitin (Ub) processing pathway. This method can be used to cleavea long polypeptide comprising two protein domains into two separateactive proteins. In this method, a single gene cassette can encode twoORFs, where the two ORFs, e.g., for crtB and EPSPS-CP4 are separated bythe 14 C-terminal amino acids of Ub, followed by a full-length Ubsequence. After translation in vivo, endogenous de-ubiquitinatingenzymes (DUBs) cleaves the polyprotein into three separate units: 1) theN-terminal protein, which comprises the identification sequence crtBterminating in the 14 C-terminal amino acids of Ub; 2) a Ub monomer; and3) the C-terminal polypeptide, which encodes a selectable markerEPSPS-CP4. Such methods are known to those skilled in the art (e.g.Walker et al, 2007).

A transcriptional fusion between an identification sequence and aselectable or a screenable marker can be made by using internal ribosomeentry sites (IRES). For example, a transcript could be made that encodesthe ORF of crtB followed by the ORF of ESPSP-CP4 with a functional IRESelement positioned between them. Several IRES are known to those skilledin the art (see e.g., Dinkova et al. 2005 and references therein; andU.S. Pat. No. 7,119,187, incorporated herein by reference).

The phenotype of the identification sequence in seed tissue may also bedetected by methods including visual, biochemical, immunological andnucleic-acid based (e.g. PCR-based) methods, among others. Theidentification sequence may confer a detectable phenotype in seed tissuethat may be distinguished from the phenotype of the marker gene. Thephenotype conferred by the identification sequence can include alteredseed germination. The identification sequence may be expressed in one ormore portions of a seed (kernel), including the embryo, endosperm,cotylcdon(s), and seed coat (testa), such that a phenotype may bediscerned.

The identification sequence may also cause a seedless phenotype. Toaccomplish tissue ablation, in one embodiment, the identificationsequence directs ovule-specific expression of defH9-iaaM or rolB inplants (e.g. Rotino et al. 1997, Carmi et al. 2003, GenBank AM422760,X64255, AE009418), which abolishes ovule development and results in aseedless phenotype in marker and identification sequence-containingovaries. In yet another embodiment, the identification sequence directsover expression of OsCDPK2 in cereal crops disrupting seed development(Morello et al. 2000; e.g., GenBank Y13658).

Genetic elements may also be designed to suppress the expression of anendogenous gene, resulting in the production of a seed phenotype thatpermits distinguishing of seeds that contain the marker gene from thosethat do not. The genetic elements of this identification sequence arephysically linked to the marker gene, e.g. embedded within the markerDNA cassette, such that the seed phenotype is linked to the presence ofthe marker, allowing for the rapid identification of marker containingseeds.

RNAi may be used to silence one or more genes resulting in an easilyscored, preferably visible, seed phenotype. The DNA sequences requiredfor an RNAi-mediated seed phenotype are positioned on the same T-DNA asthe marker gene. Any progeny seed that contains the marker gene wouldalso display the seed phenotype and would be easily identified. Suchseeds would not need to be grown and screened for the presence of themarker gene. Thus, only seeds without the phenotype conferred by theidentification sequence are grown and/or screened for the presence ofthe GOI. Depending on whether the seeds are from self-pollination oroutcrossing, this method reduces the number of seeds that need to beplanted and screened by at least 3× for selfing plant species or 1× foroutcrossing plant species.

A wide variety of compositions are known to those skilled in the artthat can be used to silence a target gene using RNAi related pathways.One embodiment is to assemble a DNA cassette that will transcribe aninverted repeat of sequences, to produce a double-stranded RNA (dsRNA),typically at least about 19-21 bp in length and corresponding to aportion of one or more genes targeted for silencing. The dsRNA can beabout 19-21 bp in length and corresponding to a portion of one or moregenes targeted for silencing. This DNA cassette including anidentification sequence is positioned within the same T-DNA as theselectable marker gene. Other methods to silence a gene known to thoseskilled in the art include, but are not limited to: cosuppression,antisense, expression of miRNAs (natural or engineered), expression oftrans-acting siRNAs, and expression of ribozymes. Any of these methodsmay be used if the sequences required for the gene silencing effect arepositioned in the same T-DNA as the marker gene.

The identification sequence may increase or decrease seed size. In oneembodiment, the identification sequence confers down regulation of AP2gene (e.g., GenBank U12546) by antisense RNA or RNA interference orcosuppression technology (Jofuku et al., 2005), which results in largerseeds containing the identification sequence and selectable markergenes. The larger seed size may also be achieved by ectopic expressionof an AFR2 gene (Schruff et al., 2006; e.g., GenBank AccessionsNM_(—)203251; NM_(—)180913), or ANT transcription factor (Mizukami andFisher, 2000; e.g., GenBank NM_(—)202701, NM_(—)119937, NM_(—)180024,NM_(—)101474, NM_(—)202110). In another embodiment, the identificationsequence conveys down regulation of a LEC2 (e.g., GenBank AF400123) or aSnf-1 (GenBank AB101657, AB101656, AB101655) gene by antisense RNA orRNAi or cosuppression technology, which leads to decreased seed size(Mendoza et al., 2005; Radchuk et al., 2006). The changed seed size canbe easily sorted by weight, shape or sieving by manual and/or mechanicalmeans.

It is specifically contemplated that automated screening techniques maybe implemented with the current invention for the identification ofseeds having a particular detectable phenotype. In this manner largenumbers of seeds can be efficiently screened and seeds lacking anidentification sequence may be collected. Automated techniques may befaster, less expensive and more accurate than reliance upon humantechnicians. Such seed sorting machines which could be used in thismanner have been described. For example, U.S. Pat. No. 4,946,046describes an apparatus for sorting seeds according to color. In thismachine, seeds are sorted according to color by placing the seeds inuniform rows of indentations in a rotating drum and passing the seedsbeneath a digital imaging camera and a light source. Images are read bythe camera and are fed to a computer, which also receives informationfrom a drum speed sensor. The computer generates a signal which causes ablast of air to blow through an opening in the bottom of an indentationcontaining a colored seed to collect such seed. Collected seeds are fedinto a collection hopper, and the non-colored seeds into a separatehopper.

By varying the wavelength of the light source used for detection ofcolored seeds, as well as barrier filters placed between the coloredseed and the detection camera, potentially any identification markercould be detected with this technique. For example, to detect seedsexpressing GFP, the excitation wavelength is in the blue light UVspectrum, typically at about 395 nm. Suitable light sources for UVemission are well known to those of skill in the art, and include xenonor mercury lamps. Suitable filter sets also are well known to those ofskill in the art, and include, for example, a BP450-490 exciter filter,an FT510 chromatic beam splitter, and a BP515-565 barrier filter (CarlZeiss, Inc., Thomwood, N.Y.). Such filter sets and emission wavelengthsare discussed in more detail in Heim and Tsien, 1996, the disclosure ofwhich is specifically incorporated herein by reference in its entirety.

By use of constructs including one or more identification sequence(s),the selective power can be extended to multiple selectable and/orscreenable genes and genes of interest. Therefore, large numbers oftransgenic seeds, representing a variety of different transformationevents, can be efficiently screened and only those seeds having (orlacking) a desired set of identification sequences may be selected.

A recombinant DNA vector may, for example, be a linear DNA segment or aclosed circular plasmid. The vector system may be a single vector orplasmid or two or more vectors or plasmids that together contain thetotal DNA to be introduced into the plant genome. Nucleic acid moleculesas set forth herein can, for example, be suitably inserted into a vectorunder the control of a suitable promoter that functions in a plant cellto drive expression of a linked coding sequence or other DNA sequence.Many vectors are available for this purpose, and selection of theappropriate vector will depend mainly on the size of the nucleic acid tobe inserted into the vector and the particular host cell to betransformed with the vector. Each vector contains various componentsdepending on its function and the particular vector and plant cell withwhich it is used or is compatible.

A number of vectors suitable for stable transformation of plant cells orfor the establishment of transgenic plants are well known, e.g., Gelvinet al. (1990). Typically, plant expression vectors include, but are notlimited to, one or more gene of interest transcription units, each ofwhich includes: a 5′ untranslated region, which includes sequences thatcontrol transcription (e.g., cis-acting promoter sequences such asenhancers, the transcription initiation start site, etc.) andtranslation (e.g., a ribosome binding site) of an operably linkedprotein-coding sequence (“open reading frame”, ORF); a 3′ untranslatedregion that includes additional regulatory regions from the 3′ end ofplant genes (Thornburg et al., 1987); An et al., 1989), e.g., a 3′terminator region to increase mRNA stability. Alternatively a plantexpression vector may be designed for expression of an mRNA moleculethat may, for instance, alter plant gene expression by an RNAi-mediatedapproach. In addition, such constructs commonly include a selectableand/or screenable marker transcription unit and optionally an origin ofreplication or other sequences required for replication of the vector ina bacterial host cell.

The constructs may also contain the plasmid backbone DNA segments thatprovide replication function and antibiotic selection in bacterialcells, for example, an Escherichia coli origin of replication such asori322, a broad host range origin of replication such as oriV or oriRi,and a coding region for a selectable marker such as Spec/Strp thatencodes for Tn7 aminoglycoside adenyltransferase (aadA) conferringresistance to spectinomycin or streptomycin, or a gentamicin (Gm, Gent)selectable marker gene. For plant transformation, the host bacterialstrain is often Agrobacterium tumefaciens ABI, C58, LBA4404, EHA101, orEHA105 carrying a plasmid having a transfer function for the expressionunit. Other strains known to those skilled in the art of planttransformation can function in the present invention.

Plant expression vectors optionally include RNA processing signals,e.g., introns, which may be positioned upstream or downstream of apolypeptide-encoding sequence in the transgene. In addition, theexpression vectors may also include additional regulatory sequences fromthe 3′-untranslated region of plant genes. These 3′ untranslated regionscontain mRNA transcription termination signals. Other movable elementscontained in plant expression vectors may include 5′ leader sequences,transit signal sequences, and coding sequences.

Expression and cloning vectors may contain a selection gene, alsoreferred to as a plant selectable marker. This gene encodes a proteinnecessary for the survival or growth of transformed plant cells grown ina selective culture regimen. Typical selection genes encode proteinsthat confer resistance to selective agents such as antibiotics includingherbicides, or other toxins, e.g., neomycin, methotrexate, dicamba,glufosinate, or glyphosate. Those cells that are successfullytransformed with a heterologous protein or fragment thereof produce aprotein conferring, e.g. drug resistance and thus survive the selectionregimen. Examples of various selectable/screenable/scorable markers andgenes encoding them are disclosed in Miki and McHugh, 2004.

An expression vector for producing a mRNA can also contain an inducibleor tissue specific promoter that is recognized in the host plant celland is operably linked to the nucleic acid encoding, the nucleic acidmolecule, or fragment thereof, of interest. Plant promoters arediscussed below.

In one embodiment, the plant transformation vector that is utilizedincludes an isolated and purified DNA molecule including a heterologousseed-specific promoter operatively linked to one or more nucleotidesequences of the present invention. In another embodiment, the promoteris seed-expressed, but not seed-specific. A plant transformation vectormay contain sequences from one or more genes, thus allowing productionof more than mRNA in a plant cell. One skilled in the art will readilyappreciate that segments of DNA can be combined into a single compositeDNA segment for expression in a transgenic plant.

Suitable methods for transformation of host cells for use with thecurrent invention are believed to include virtually any method by whichDNA can be introduced into a cell (see, for example, Miki et al., 1993),such as by transformation of protoplasts (U.S. Pat. No. 5,508,184;Omirulleh et al., 1993), by desiccation/inhibition-mediated DNA uptake(Potrykus et al., 1985), by electroporation (U.S. Pat. No. 5,384,253),by agitation with silicon carbide fibers (Kaeppler et al., 1990; U.S.Pat. No. 5,302,523; and U.S. Pat. No. 5,464,765), byAgrobacterium-mediated transformation (U.S. Pat. Nos. 5,563,055;5,591,616; 5,693,512; 5,824,877; 5,981,840; 6,384,301) and byacceleration of DNA coated particles (U.S. Pat. Nos. 5,015,580;5,550,318; 5,538,880; 6,160,208; 6,399,861; 6,403,865; Padgette et al.1995), etc. Through the application of techniques such as these, thecells of virtually any species may be stably transformed. In the case ofmulticellular species, the transgenic cells may be regenerated intotransgenic organisms.

The most widely utilized method for introducing an expression vectorinto plants is based on the natural transformation system ofAgrobacterium (for example, Horsch et al., 1985) The Ti and Ri plasmidsof A. tumefaciens and A. rhizogenes, respectively, carry genesresponsible for genetic transformation of the plant. Descriptions ofAgrobacterium vector systems and methods for Agrobacterium-mediated genetransfer are provided by numerous references, including Gruber et al.,1993; Miki et al., 1993, Moloney et al., 1989, and U.S. Pat. Nos.4,940,838 and 5,464,763. Other bacteria such as Sinorhizobium,Rhizobium, and Mesorhizobium that interact with plants naturally can bemodified to mediate gene transfer to a number of diverse plants(Broothaerts et al., 2005). These plant-associated symbiotic bacteriacan be made competent for gene transfer by acquisition of both adisarmed Ti plasmid and a suitable binary vector. DNA sequences to betransferred via an Agrobacterium-mediated transformation method includeone or more “border” sequences, such as right border (RB) and leftborder (LB) sequences that usually define the extent of the transferredDNA (T-DNA) containing one or more genes to be expressed in a plantcell, and may further include an enhancer sequence such as an overdrivesequence (Toro et al., 1989) or a plurality of overdrive sequences asdisclosed in U.S. Provisional Patent Application No. 60/831,814,incorporated herein by reference.

Techniques that may be particularly useful in the context of cottontransformation are disclosed in U.S. Pat. Nos. 5,846,797, 5,159,135,5,004,863, and 6,624,344. Techniques for transforming Brassica plants inparticular are disclosed, for example, in U.S. Pat. No. 5,750,871; andtechniques for transforming soybean are disclosed in, for example, Zhanget al., 1999, U.S. Pat. No. 6,384,301, and U.S. Pat. No. 7,002,058.Techniques for transforming corn are disclosed in WO9506722. Somenon-limiting examples of plants that may find use with the inventioninclude alfalfa, barley, beans, beet, broccoli, cabbage, carrot, canola,cauliflower, celery, Chinese cabbage, corn, cotton, cucumber, dry bean,eggplant, fennel, garden beans, gourd, leek, lettuce, melon, oat, okra,onion, pea, pepper, pumpkin, peanut, potato, pumpkin, radish, rice,sorghum, soybean, spinach, squash, sweet corn, sugarbeet, sunflower,tomato, watermelon, and wheat.

A vector or construct may also include various regulatory elements. The5′ non-translated leader sequence can be derived from the promoterselected to express the heterologous gene sequence of the DNA moleculeof the present invention, and can be specifically modified if desired soas to increase translation of mRNA. The 5′ non-translated regions canalso be obtained from plant viral RNAs (e.g. Tobacco mosaic virus,Tobacco etch virus, Maize dwarf mosaic virus, Alfalfa mosaic virus) fromsuitable eukaryotic genes, plant genes (wheat and maize chlorophyll a/bbinding protein gene leader), or from a synthetic gene sequence. Theleader sequence could also be derived from an unrelated promoter orcoding sequence. Leader sequences useful in context of the presentinvention include the maize Hsp70 leader (U.S. Pat. No. 5,362,865 andU.S. Pat. No. 5,859,347, herein incorporated by reference in theirentirety), and the TMV omega element (Gallie et al., 1989). Examples oftranslation leader sequences include maize and petunia heat shockprotein leaders (U.S. Pat. No. 5,362,865), plant virus coat proteinleaders, plant rubisco leaders, GmHsp (U.S. Pat. No. 5,659,122), PhDnaK(U.S. Pat. No. 5,362,865), AtAnt1, TEV (Carrington and Freed, 1990),AGRtunos (GenBank Accession V00087; Bevan et al., 1983), OsAct1 (U.S.Pat. No. 5,641,876), OsTPI (U.S. Pat. No. 7,132,528), and OsAct15 (USPublication No. 20060162010), among others.

Intron sequences are known in the art to aid in the expression oftransgenes in monocot plant cells. Examples of introns include the cornactin intron (U.S. Pat. No. 5,641,876), the corn HSP70 intron (ZmHSP70;U.S. Pat. No. 5,859,347; U.S. Pat. No. 5,424,412), and rice TPI intron(OsTPI; U.S. Pat. No. 7,132,528), and are of benefit in practicing thisinvention.

A vector may also include a transit peptide nucleic acid sequence. Manychloroplast-localized proteins, including those involved in carotenoidsynthesis, are expressed from nuclear genes as precursors and aretargeted to the chloroplast by a chloroplast transit peptide (CTP) thatis removed after the import steps. Examples of other such chloroplastproteins include the small subunit (SSU) of Ribulose-1,5,-bisphosphatecarboxylase, and the light-harvesting complex protein I and protein II.It has been demonstrated in vivo and in vitro that non-chloroplastproteins may be targeted to the chloroplast by use of protein fusionswith a CTP and that a CTP sequence is sufficient to target a protein tothe chloroplast. Incorporation of a suitable chloroplast transitpeptide, such as the Arabidopsis thaliana (At) EPSPS CTP (Klee et al.,1987), and the Petunia hybrida (Ph.) EPSPS CTP (della-Cioppa et al.,1986) has been shown to target heterologous protein sequences tochloroplasts in transgenic plants. Those skilled in the art willrecognize that various chimeric constructs can be made, if needed, thatutilize the functionality of a particular CTP to import a given geneproduct into a chloroplast. Other CTPs that may be useful in practicingthe present invention include PsRbcS-derived CTPs (Pisum sativum Rubiscosmall subunit CTP; Coruzzi et al., 1984); AtRbcS CTP (Arabidopsisthaliana Rubisco small subunit 1A CTP; CTP1; U.S. Pat. No. 5,728,925);AtShkG CTP (CTP2; Klee et al., 1987); AtShkGZm CTP (CTP2synthetic; codonoptimized for monocot expression; SEQ ID NO:14 of WO04009761); PhShkGCTP (Petunia hybrida EPSPS; CTP4; codon optimized for monocotexpression; Gasser et al., 1988); TaWaxy CTP (Triticum aestivumgranule-bound starch synthase CTPsynthetic, codon optimized for cornexpression: Clark et al., 1991): OsWaxy CTP (Oryza sativa starchsynthase CTP; Okagaki, 1992); NtRbcS CTP (Nicotiana tabacum ribulose1,5-bisphosphate carboxylase small subunit chloroplast transit peptide;Mazur, et al., 1985); ZmAS CTP (Zea mays anthranilate synthase alpha 2subunit gene CTP; Gardiner et al., 2004); and RgAS CTP (Ruta graveolensanthranilate synthase CTP; Bohlmann, et al., 1995). Other transitpeptides that may be useful include maize cab-m7 signal sequence (PCT WO97/41228) and the pea (Pisum sativum) glutathione reductase signalsequence (PCT WO 97/41228).

Termination of transcription may be accomplished by a 3′ non-translatedDNA sequence operably linked to a recombinant transgene (e.g. the geneof interest, the identification sequence including a screenable gene, orthe plant selectable marker gene). The 3′ non-translated region of arecombinant DNA molecule contains a polyadenylation signal thatfunctions in plants to cause the addition of adenylate nucleotides tothe 3′ end of the RNA. The 3′ non-translated region can be obtained fromvarious genes that are expressed in plant cells. The nopaline synthase3′ untranslated region (Fraley et al., 1983), is commonly used in thiscapacity. Polyadenylation molecules from a Pisum sativuin RbcS2 gene(Ps.RbcS2-E9; Coruzzi et al., 1984), AGRtu.nos (Genbank AccessionE01312), E6 (Accession # U30508), rice glutelin (Okita et al., 1989),and TaHsp17 (wheat low molecular weight heat shock protein gene;Accession # X13431) in particular may be of benefit for use with theinvention.

For embodiments of the invention in which the use of a constitutivepromoter is desirable, any well-known constitutive plant promoter may beused. Constitutive plant promoters include, for example, the cauliflowermosaic virus (CaMV) 35S promoter, which confers constitutive, high-levelexpression in most plant tissues (see, e.g., Odell et al., 1985),including monocots (see, e.g., Dekeyser et al., 1990); Terada et al.,1990); the nopaline synthase promoter (An et al., 1988), the octopinesynthase promoter (Fromm et al., 1989), cauliflower mosaic virus 19Spromoter, figwort mosaic virus 35S promoter, rice actin 1 promoter,mannopine synthase promoter, and a histone promoter.

For other embodiments of the invention, well-known plant gene promotersthat are regulated in response to environmental, hormonal, chemical,and/or developmental signals may be used, including promoters regulatedby (1) heat (Callis et al., 1988), (2) light (e.g., pea rbcS-3Apromoter, Kuhlemeier et al., 1989; maize rbcS promoter, Schaffner andSheen, 1991; or chlorophyll a/b-binding protein promoter, Simpson etal., 1985), (3) hormones, such as abscisic acid (Marcotte et al., 1989),(4) wounding (e.g., wunl, Siebertz et al., 1989); or (5) chemicals suchas methyl jasmonate, salicylic acid, etc. It may also be advantageous toemploy (6) organ-specific promoters (e.g., Roshal et al., 1987;Schemthaner et al., 1988; Bustos et al., 1989).

There are a wide variety of plant promoter sequences which may be usedto drive tissue-specific expression of polynucleotides in transgenicplants. Indeed, in particular embodiments of the invention, the promoterused is a seed specific promoter. The promoter for β-conglycinin (Chenet al., 1989) or other seed-specific promoters such as the napinpromoter, which are regulated during plant seed maturation (Kridl etal., 1991; Kohno-Murase et al., 1994), barley Hv.Perl (Stacey et al.,1996), phaseolin (Bustos et al., 1989), soybean trypsin inhibitor (Riggset al., 1989), ACP (Baerson et al., 1993), stearoyl-ACP desaturase(Slocombe et al., 1994), soybean α′ subunit of β-conglycinin (P-Gm7S,see for example, Chen et al., 1986), Vicia faba USP (P-Vf.Usp, see forexample, SEQ ID NO: 1, 2, and 3, U.S. Appln. Pub. 20030229918), theglobulin promoter (see for example Belanger and Kriz, 1991), soybeanalpha subunit of β-conglycinin (7S alpha; U.S. Pat. No. 6,825,398,incorporated by reference) and Zea maysL3 oleosin promoter (P-Zm.L3,see, for example, Hong et al., 1997; see also U.S. Pat. No. 6,433,252,the disclosure of which is specifically incorporated herein byreference).

The zeins are a group of storage proteins found in Zea mays endosperm.Genomic clones for zein genes have been isolated (Pedersen et al., 1982;U.S. Pat. No. 6,326,527), and the promoters from these clones, includingthe 15 kDa, 16 kDa, 19 kDa, 22 kD, 27 kDa, and gamma genes, could alsobe used. Other promoters known to function, for example, in Zea maysinclude the promoters for the following genes: waxy (Russell and Fromm,1997; Shure et al., 1983), Brittle (Giroux et al., 1994), Shrunken 2,Branching enzymes I and II, starch synthases, debranching enzymes,oleosins, glutelins, and sucrose synthases. Another promoter for Zeamays endosperm expression is the promoter for the glutelin gene fromrice, more particularly the Osgt-1 promoter (Zheng et al., 1993).Examples of such promoters in rice include those promoters for theADPGPP subunits, the granule bound and other starch synthase, thebranching enzymes, the debranching enzymes, sucrose synthases (Yang etal., 1990), and Betl1 (basal endosperm transfer layer) and globulin1.

Examples of other promoters that may be useful with the presentinvention are described in the U.S. Pat. No. 6,437,217 (maize RS81promoter), U.S. Pat. No. 5,641,876 (rice actin promoter; OsAct1), U.S.Pat. No. 6,426,446 (maize RS324 promoter), U.S. Pat. No. 6,429,362(maize PR-1 promoter), U.S. Pat. No. 6,232,526 (maize A3 promoter), U.S.Pat. No. 6,177,611 (constitutive maize promoters), U.S. Pat. Nos.5,322,938, 5,352,605, 5,359,142 and 5,530,196 (35S promoter), U.S. Pat.No. 6,433,252 (maize L3 oleosin promoter), U.S. Pat. No. 6,429,357 (riceactin 2 promoter as well as a rice actin 2 intron), U.S. Pat. No.5,837,848 (root specific promoter), U.S. Pat. No. 6,294,714 (lightinducible promoters), U.S. Pat. No. 6,140,078 (salt induciblepromoters), U.S. Pat. No. 6,252,138 (pathogen inducible promoters), U.S.Pat. No. 6,175,060 (phosphorus deficiency inducible promoters), U.S.Pat. No. 6,635,806 (gamma-coixin promoter), and U.S. Pat. No. 7,151,204(maize chloroplast aldolase promoter). Additional promoters that mayfind use are a nopaline synthase (NOS) promoter (Ebert et al., 1987),the octopine synthase (OCS) promoter (which is carried on tumor-inducingplasmids of Agrobacterium tumefaciens), the caulimovirus promoters suchas the cauliflower mosaic virus (CaMV) 19S promoter (Lawton et al.,1987), the CaMV 35S promoter (Odell et al., 1985), the figwort mosaicvirus 35S-promoter (Walker et al., 1987), the sucrose synthase promoter(Yang et al., 1990), the R gene complex promoter (Chandler et al.,1989), and the chlorophyll alb binding protein gene promoter, etc. Inthe present invention, CaMV35S with enhancer sequences (e35S; U.S. Pat.Nos. 5,322,938; 5,352,605; 5,359,142; and 5,530,196), FMV35S (U.S. Pat.Nos. 6,051,753; 5,378,619), peanut chlorotic streak caulimovirus (PClSV;U.S. Pat. No. 5,850,019), At.Act 7 (Accession # U27811), At.ANT1 (USPatent Application Publication 20060236420), FMV.35S-EF1a (U.S. PatentApplication Publication 20050022261), eIF4A10 (Accession # X79008) andAGRtu.nos (GenBank Accession V00087; Depicker et al, 1982; Bevan et al.,1983), rice cytosolic triose phosphate isomerase (OsTPI; U.S. Pat. No.7,132,528), and rice actin 15 gene (OsAct15; U.S. Patent ApplicationPublication 20060162010) promoters may be of particular benefit. In someinstances, e.g., OsTPI and OsAct 15, a promoter may include a 5′UTRand/or a first intron. Other promoters useful in the practice of theinvention that are known by one of skill in the art are alsocontemplated by the invention.

A plant expression vector may also include a screenable or scorablemarker gene cassette that may be used in the present invention tomonitor segregating cells or progeny for (loss of) expression. Exemplarymarkers are known and include β-glucuronidase (GUS) that encodes anenzyme for various chromogenic substrates (Jefferson et al., 1987a;Jefferson et al., 1987b); an R-locus gene, that encodes a product thatregulates the production of anthocyanin pigments (red color) in planttissues (Dellaporta et al., 1988); a β-lactamase gene (Sutcliffe et al.,1978); a gene that encodes an enzyme for that various chromogenicsubstrates are known (e.g., PADAC, a chromogenic cephalosporin); aluciferase gene (Ow et al., 1986); a xylE gene (Zukowsky et al., 1983)that encodes a catechol dioxygenase that can convert chromogeniccatechols; an α-amylase gene (Ikatu et al., 1990); a tyrosinase gene(Katz et al., 1983) that encodes an enzyme capable of oxidizing tyrosineto DOPA and dopaquinone that in turn condenses to melanin; greenfluorescence protein (Elliot et al., 1999) and an α-galactosidase. Ascreenable or scorable marker gene may encode the same gene product asan identification sequence including a screenable or scorable gene, or adifferent gene product. However, the identification sequence isexpressed in egg, pollen or seed tissues, while the screenable orscorable marker gene is expressed during the process of identifyingtransformed plant cells. The identification sequence may also beexpressed constitutively, but only convey a phenotype in egg, pollen, orseed tissues.

Transgenic plants may be regenerated from a transformed plant cell bymethods well known in the field of plant cell culture. A transgenicplant formed using Agrobacterium transformation methods typicallycontains a single simple recombinant DNA sequence inserted into onechromosome and is referred to as a transgenic event. Such transgenicplants can be referred to as being heterozygous for the insertedexogenous sequence. A transgenic plant homozygous with respect to atransgene can be obtained by sexually mating (selfing) an independentsegregant transgenic plant that contains a single exogenous genesequence to itself, for example an F0 plant, to produce F1 seed. Onefourth of the F1 seed produced will be homozygous with respect to thetransgene. Germinating F1 seed results in plants that can be tested forzygosity, typically using a SNP assay or a thermal amplification assaythat allows for the distinction between heterozygotes and homozygotes(i.e., a zygosity assay).

A number of identification sequences may be used, for instance geneswhose expression may result in a visible phenotype, including use ofgus, gfp, and luc (see, e.g., Ow et al., 1986; WO 97/41228 and U.S. Pat.No. 6,583,338; e.g., M26194; M15077). A levansucrase gene, sacB (Caimiet al., 1996; e.g., X02730) leading to a “shrunken” seed phenotype, or apyrophosphatase gene (Hajirezaei et al., 1999) leading to inhibition ofgermination, may also be employed. Genes encoding phytoene synthase(crtB) are known in the art, including those from Erwinia uredovora(e.g. Misawa et al., 1990; Sandmann and Misawa, 1992; U.S. Pat. Nos.5,429,939; 6,429,356), and Pantoea/Enterobacter agglomerans (e.g.GenBank M38423; M87280), among others. Seed-specific expression of crtBthat results in orange coloration has been described (Shewmaker et al.,1999; U.S. Pat. No. 6,429,356).

Most transgenes producing pleiotropic seed phenotypes may be used as avisible label gene linked to a selectable marker to identify marker-freegene of interest positive seeds. The visible phenotype may be producedby ectopic overexpression of a transgene or result from down regulationof endogenous metabolic pathway genes by antisense RNA, RNA interferenceor co-suppression technology.

The following definitions and methods are provided to better define thepresent invention and to guide those of ordinary skill in the art in thepractice of the present invention. Unless otherwise noted, terms are tobe understood according to conventional usage by those of ordinary skillin the relevant art. Definitions of common terms in molecular biologymay also be found in Rieger et al. (1991); and Lewin (1994). Thenomenclature for DNA bases as set forth at 37 CFR §1.822 is used.

“CP4”, “aroA:CP4”, “AGRTU.aroA:CP4”, “CP4 EPSPS” and “EPSPS CP4” referto the EPSP synthase gene or protein purified from Agrobacteriumtumefaciens (AGRTU) strain CP4 that when expressed in plants conferstolerance to glyphosate and glyphosate containing herbicide formulations(U.S. Pat. No. 5,633,435, herein incorporated by reference in itsentirety). The gene sequence may be native or modified for enhancedexpression in plants.

A DNA “segment” refers to a region of DNA sequence of a DNA construct. ADNA segment may be within, between, or flanking the T-DNA moleculesfound in a construct used for Agrobacterium-mediated plant celltransformation. For instance, a DNA segment may contain genetic elementsfor replication of plasmids in bacteria or other various elements andexpression cassettes of the DNA construct designed for use in plant celltransformation. Thus, a “DNA cassette” may comprise a DNA segment,including element(s) for expression of the DNA sequence in a cell.

A “fusion protein” refers to a translational fusion expressed as asingle unit, yet producing a gene product conferring the phenotypes ofthe protein encoded by the non-fused starting gene sequences.

An “isolated” nucleic acid is substantially separated or purified awayfrom other nucleic acid sequences in the cell of the organism in whichthe nucleic acid naturally occurs, i.e., other chromosomal andextrachromosomal DNA and RNA, by conventional nucleic acid-purificationmethods. The term also embraces recombinant nucleic acids and chemicallysynthesized nucleic acids.

The term “glyphosate resistance gene” refers to any gene that, whenexpressed as a transgene in a plant, confers the ability to toleratelevels of the herbicide glyphosate that would otherwise damage or killthe plant. Any glyphosate tolerance gene known to the skilled individualare suitable for use in the practice of the present invention.Glyphosate (including any herbicidally active form ofN-phosphonomethylglycine and any salt thereof) inhibits the enzyme5-enolpyruvylshikimate-3-phosphate synthase (EPSPS). A variety of nativeand variant EPSPS enzymes have been expressed in transgenic plants inorder to confer glyphosate tolerance, any of which can be used in theinvention. Examples of some of these EPSPSs include those describedand/or isolated in accordance with U.S. Pat. No. 4,940,835, U.S. Pat.No. 4,971,908, U.S. Pat. No. 5,145,783, U.S. Pat. No. 5,188,642, U.S.Pat. No. 5,310,667, and U.S. Pat. No. 6,803,501. They can also bederived from a structurally distinct class of non-homologous EPSPSgenes, such as the class II EPSPS genes isolated from Agrobacterium sp.strain CP4 (AGRTU.aroA:CP4).

The term “identification sequence” refers to a nucleic acid that encodesa product conferring a detectable phenotype such as a change in seed orgamete color, opacity or translucence, fluorescence, texture, size,shape, germinability, or viability, or other product of cell or seedmetabolism. The identification sequence may include a nucleotidesequence (e.g. a gene fragment) that may confer a phenotype via downregulation of the expression of another gene, such as via anRNAi-mediated process. In certain embodiments, the identificationsequence includes a screenable gene such as a gusA, gfp, or crtB gene.In a particular embodiment, the identification sequence includes a crtBgene encoding a phytoene synthase from Erwinia herbicola (Pantoeaagglomerans; GenBank M38423, incorporated herein by reference; and U.S.Pat. Nos. 5,429,939, 6,429,356). The identification sequence isphysically linked to a plant selectable, screenable, and/or scorablemarker gene, such as one encoding antibiotic resistance or herbicidetolerance. The identification sequence can confer a detectable (e.g.screenable or selectable) phenotype in seed.

A first nucleic-acid sequence is “operably” connected or “linked” with asecond nucleic acid sequence when the first nucleic acid sequence isplaced in a functional relationship with the second nucleic acidsequence. For instance, a promoter is operably linked to aprotein-coding sequence if the promoter affects the transcription orexpression of the coding sequence. Generally, operably linked DNAsequences are contiguous and, where necessary to join two protein-codingregions, are in the same reading frame.

The term “plant” encompasses any higher plant and progeny thereof,including monocots (e.g., lily, corn, rice, wheat, barley, etc.), dicots(e.g., soybean, cotton, tomato, canola, potato, Arabidopsis, tobacco,etc.), gymnosperms (pines, firs, cedars, etc.) and includes parts ofplants, including reproductive units of a plant (e.g., seeds, bulbs,tubers, or other parts or tissues from that the plant can bereproduced), fruit, flowers, etc.

A “recombinant” nucleic acid is made by an artificial combination of twootherwise separated segments of sequence, e.g., by chemical synthesis orby the manipulation of isolated segments of nucleic acids by geneticengineering techniques.

The terms “DNA construct” or “DNA vector” refers to any plasmid, cosmid,virus, autonomously replicating sequence, phage, or other circularsingle-stranded or double-stranded DNA or RNA derived from any sourcethat includes one or more DNA sequences, such as promoters,protein-coding sequences, 3′ untranslated regions, etc., that have beenlinked in a functionally operative manner by recombinant DNA techniques.Recombinant DNA vectors for plant transformation are commonlydouble-stranded circular plasmids capable of replication in a bacterialcell. Conventional compositions and methods for making and usingrecombinant nucleic acid constructs are well known, e.g. Sambrook etal., 1989; and Ausubel et al., 1992 (with periodic updates), and Clarket al. (1997), among others.

The term “promoter” or “promoter region” refers to a nucleic acidsequence, usually found upstream (5′) to a coding sequence that controlsexpression of the coding sequence by controlling production of messengerRNA (mRNA) by providing the recognition site for RNA polymerase and/orother factors necessary for start of transcription at the correct site.As contemplated herein, a promoter or promoter region includesvariations of promoters derived by means of ligation to variousregulatory sequences, random or controlled mutagenesis, and addition orduplication of enhancer sequences. A promoter region is responsible fordriving the transcription of coding sequences under their control whenintroduced into a host as part of a suitable recombinant vector, asdemonstrated by its ability to produce mRNA.

“Regeneration” refers to the process of growing a plant from a plantcell (e.g., plant protoplast or explant).

“Selectable marker” refers to a nucleic acid sequence whose expressionconfers a phenotype facilitating identification of cells containing thenucleic acid sequence. Selectable markers include those that conferresistance to toxic chemicals (e.g. antibiotic resistance), or impart avisually distinguishing characteristic (e.g. color changes orfluorescence).

Useful dominant plant selectable marker genes include genes encodingantibiotic resistance genes (e.g. resistance to hygromycin,imidazolinone, kanamycin, bleomycin, G418, streptomycin orspectinomycin); and herbicide resistance genes (e.g. phosphinothricinacetyltransferase, modified ALS, BAR, modified class I EPSPSs, class IIEPSPSs, DMOs), among others.

Included within the terms “storable marker genes” or “screenable markergenes” are genes that encode a secretable marker whose secretion can bedetected as a means of identifying or selecting for transformed cells.Examples include markers that encode a secretable antigen that can beidentified by antibody interaction, or even secretable enzymes that canbe detected catalytically. Secretable proteins fall into a number ofclasses, including small, diffusible proteins that are detectable,(e.g., by ELISA), small active enzymes that are detectable inextracellular solution (e.g. α-amylase, β-lactamase, phosphinothricinacetyltransferase), or proteins that are inserted or trapped in the cellwall (such as proteins that include a leader sequence such as that foundin the expression unit of extension or tobacco PR-S). Other possibleselectable and/or screenable marker genes will be apparent to those ofskill in the art.

“T-DNA” refers to a DNA molecule that integrates into a plant genome viaan Agrobacterium or other Rhizobia-mediated transformation method. Atleast one end of the T-DNA molecule is flanked by at least one borderregion of the T-DNA from an Agrobacterium Ti or Ri plasmid. These borderregions are generally referred to as the Right border (RB) and Leftborder (LB) regions and exist as variations in nucleotide sequence andlength depending on their source (e.g. nopaline or octopine producingstrains of Agrobacterium). The border regions commonly used in DNAconstructs designed for transferring transgenes into plants are oftenseveral hundred polynucleotides in length and include a nick site wherevirD2 endonuclease derived from Ti or Ri helper plasmid digests the DNAand covalently attaches to the 5′ end after T-strand formation to guidethe T-strand integration into the genome of a plant. The T-DNAmolecule(s) generally contain one or more plant expression cassettes.

The term “transgene” refers to any nucleic acid sequence nonnative to acell or organism transformed into said cell or organism. “Transgene” mayalso refer to any endogenous sequence which is ectopically expressed bymodifying coding sequence or regulatory sequences. “Transgene” alsoencompasses the component parts of a native plant gene modified byinsertion of a nonnative or native nucleic acid sequence by directedrecombination.

EXAMPLES

Those of skill in the art will appreciate the many advantages of themethods and compositions provided by the present invention. Thefollowing examples are included to demonstrate the preferred embodimentsof the invention. It should be appreciated by those of skill in the artthat the techniques disclosed in the examples that follow representtechniques discovered by the inventors to function well in the practiceof the invention, and thus can be considered to constitute preferredmodes for its practice. However, those of skill in the art should, inlight of the present disclosure, appreciate that many changes can bemade in the specific embodiments that are disclosed and still obtain alike or similar result without departing from the spirit and scope ofthe invention. All references cited herein are incorporated herein byreference to the extent that they supplement, explain, provide abackground for, or teach methodology, techniques, or compositionsemployed herein.

Example 1 Preparation of 2T-DNA Vectors With an Identification Sequenceand Marker Gene, and a Gene-of-Interest

Two T-DNA plant expression vectors, pMON67465, pMON101338 and pMON101339(FIG. 1), were constructed according to standard molecular cloningprocedure (Sambrook et al., 1989). One T-DNA includes a CaMV 35Spromoter operably linked to an nptII gene encoding resistance tokanamycin and a CaMV 35S promoter operably linked to a GUS reportergene. The other T-DNA comprises a napin:ctp-crtB: napin 3′ cassette anda 35S:ctp:CP4 EPSPS cassette, that confers glyphosate resistance. ThecrtB in pMON67465 with oriV replication origin was driven by a 1.8 kbseed-specific napin promoter and 1 kb napin terminator from Brassicanapus. The crtB in pMON101338 with oriV replicon and in pMON101339 withpRi replication origin was driven by a shorter version of napin promoter(1 kb) and terminator (0.3 kb). The crtB gene, encoding a phytoenesynthase from Erwinia sp. confers orange color to soybean seed withoutaffecting transformation frequency (FIG. 3).

Example 2 Transformation and Regeneration of Soy Explants with pMON67465

Soybean (cv. A3244) tissues were transformed with pMON67465 (FIG. 1) viaan Agrobacterium-mediated method, essentially as previously described(U.S. Pat. No. 6,384,301, herein incorporated by reference). Briefly,hand excised soy meristem explants were co-cultivated with Agrobacteriumfor 2-4 days at 23° C., transferred onto WPM solid medium with 75 μMglyphosate selection in a PLANTCON and cultured at 28° C. under 16/8light/dark period. After two weeks, the explants were transferred tofresh WPM medium and cultured until shoot harvest. After 2 months,shoots with true trifolia were cut and cultured onto BRM rooting mediumfor 2-4 weeks. The rooted plantlets were grown in greenhouse for seedmaturation. Among 40 events analyzed, 72.5% displayed co-transformationwith both T-DNAs. 45% of the 40 events contained both nptII and gusgenes. Event A33908, containing T-DNAs from pMON67465, was furtheranalyzed.

Additional events with plasmid pMON67465 were obtained byre-transformation of the plasmid into the same cultivar A3244 and 13transgenic lines were obtained with transformation frequency of 0.22%.Seven out of 13 lines are gene of interest-positive and marker freeafter analyzing normal appearance seeds via PCR for presence or absenceof a gus or CP4 marker gene (FIG. 6). The orange seeds from R0 plantswere also analyzed by PCR for the presence of the crtB gene and CP4marker gene. All orange seeds but three were found to be positive forboth crtB and CP4 (FIG. 7), whereas none of normal appearance seeds(FIG. 7, white cells) contained CP4 or crtB genes, which indicated thecrtB phenotype is tightly linked to the CP4 selectable marker gene.

Thus, use of a 2 T-DNA construct with an identification sequenceincluding a screenable gene linked to a selectable marker gene allowsfor more efficient screening and selection of transgenic eventscontaining a gene of interest while lacking sequences encoding aselectable marker. An exemplary comparison between a linkage-Southernbased approach (“Standard 2T”) and a label-based (i.e., identificationsequence based) screen for identifying progeny seed in which a gene ofinterest and a selectable marker have independently segregated is foundin FIG. 8. Use of the identification sequence approach allows forscreening more transgenic events and more progeny of each event in orderto identify progeny useful for further analysis.

Example 3 CrtB/GUS/CP4 Expression in Soy Event A33908 and R1 ProgenySeed

Visual inspection of tissues from event A33908 (FIG. 2) indicated thatstems and young unfolded leaves displayed an orange cast. Leaf and roottissue was otherwise phenotypically normal in CrtB-expressing plants.Seed coats from seed of the R0 plant (i.e. the R1 generation) displayedslight CrtB expression, while the cotyledons of A33908-derived seeddisplayed a distinct orange color and some with a wrinkled phenotype(FIG. 3).

Twelve immature R1 seeds from event A33908 were dissected from the seedcoat and subjected to CP4-EPSPS ELISA, and CrtB and GUS-visual analyses(FIG. 4). Segregation of the CrtB, GUS, and CP4-EPSPS phenotypes wasevident. 9/12 seed were positive in all three assays. 1/12 seed was CrtBand CP4-EPSPS positive, but GUS negative, showing segregation of the twoT-DNAs of pMON67465, with loss of the gus gene. 2/12 seed were CP4-EPSPSELISA negative. Both of these seed were CrtB negative and GUS positive,thus demonstrating linkage between the identification sequence (crtB)and the selectable marker CP4-EPSPS gene, and segregation of thesetransgenic loci from the gus locus. The phenotypic ratio in segregatingseed was consistent with Mendelian segregation of two dominant loci.

Example 4 R1 Seed Visual Analysis

Mature seed from A33908 were visually analyzed for color, size, andshape (FIG. 5). A mixture of (i) marker-free normal (e.g. yellow andsmooth); (ii) orange and smooth; and (iii) orange and shrunken seed wasseen.

Example 5 PCR Analysis on GUS Positive Seed

INVADER PCR (e.g. Mein et al., 2000) was used to follow segregation ofthe CP4-EPSPS, crtB, and gus genes delivered by pMON67465 in seed oftransgenic soybean plants. Event A33908 was determined to contain asingle copy of the CP4-EPSPS marker gene and a single copy of the NPTIIgene. Segregation of orange:normal seed followed an expected 3:1 ratioin event A33908 (Table 1).

TABLE 1 Phenotype and genotype of progeny of transgenic plants Expect.3:1 Invader Invader Orange/Normal orange/normal Chi Pedigree CP4 NPT IIseed count seed count square GM 1 1 86/26 84/28 0.19 GM 2 1 98/56115.5/38.5  10.61 GM 1 1 130/72  151.5/50.5  12.20 GM 1 2 134/56 142.5/47.5  2.03 GM 2 0 62/30 69/23 2.84 GM 1 2 77/25 76.5/25.5 GM A3392 2 79/40 89.25/29.75 4.71 GM A339 2 4 57/18 56.25/18.75 0.04

Example 6 Use of ATP PFK as an Identification Sequence

For starch-rich cereal grains including corn, manipulation ofsugar/starch metabolism resulting in a phenotype of shrunken orabolished seed development may be utilized. Seed-specific expression ofsacB (Caimi et al. 1996), or seed-specific expression of yeast ATPdependent phosphofructokinase (ATP PFK; e.g. GenBank AccessionNC_(—)003423, bases 2297466 . . . 2300294) in corn ears results inabolished kernel development (FIG. 12). The construct pMON99575containing the CP4 selectable marker and ATP-PFK may be directly usedfor co-transformation with a one T-DNA construct containing a gene ofinterest by mixing cells of two Agrobacterium strains each including oneof these constructs and transforming a plant cell with the mixedbacterial culture. Alternatively, the seed-specific expressing ATP-PFKcassette may be subcloned into a 2T-DNA construct as an identificationsequence, for efficient identification of marker free seeds. Kernelscontaining this gene are extremely shrunken and do not germinate. Onlythe identification sequence-free and marker gene free kernels shownormal appearance.

Example 7 Use of Genes Involved in Porphyrin Synthesis as IdentificationSequences

S-adenosyl-L-methionine-dependent uroporphyrinogen III (uro′gen) methyltransferases (SUMT) produce bright red fluorescent porphyrinoidcompounds when overexpressed in E. coli, yeast, and CHO cells. Thisproperty has enabled visual selection of transformed E. coli colonies(Rossner & Scott 1995) and automated sorting of transformed yeast andCHO cells (Wildt & Deuschle 1999). This fluorescence is the result ofintracellular accumulation of di- and tri-methylated uro′gen(dihydrosirohydrochlorin and trimethylpyrorocorphin), both of which arecompounds found in porphyrin synthesis pathways (i.e., chlorophyll andcobalamin).

Cells transformed with cobA encoding SUMT from Propionibacteriumfreudenreichii (GenBank accession U13043; incorporated herein byreference) yield a fluorescent signal with absorbance peaks at 384 nmand 500 nm along with an emission band at 605 nm. The fluorescentporphyrinoids generated by the cobA uro′gen methyl transferase have agood spectral signature for marking plant material. Excitation at either384 or 500 nm avoids strong chlorophyll absorbance and the resulting redemission is readily detected as it has a substantial Stokes shift (fromthe 500 nm absorbance origin), but does not overlap with chlorophyllautofluorescence in the far red (Haseloff, 1999).

The carboxy terminus of the maize SUMT (GenBank D83391), ArabidopsisUpm1 (GenBank L47479), and E. coli CysG (GenBank X14202) proteins aresignificantly similar to proteins encoded by genes of P. freudenreichii(cobA), Pseudomonas denitrificans (cobA; GenBank M59236), and ofSynechocystis sp. (formerly Anacystis nidulans; GenBank X70966), eachincorporated herein by reference (Sakakibara et al. 1996), and may beused similarly.

A construct including a promoter with kernel expression and a geneencoding CobA, or a similar protein with SUMT activity, allows the useof such a gene as an identification sequence by screening for (lack of)visible red fluorescence in corn seed, for instance. Plant sirohemesynthases have been reported to be localized in the chloroplast (Leusteket al., 1997). Thus use of a porphyrin biosynthesis gene as anidentification sequence may include use of a chloroplast transit peptideto direct the gene product to the chloroplast. The construct can bedirectly used as an identification sequence, and a T-DNA comprising suchan identification sequence and a selectable marker may, for instance, beco-transformed with a second construct comprising a T-DNA containing agene of interest by mixing two Agrobacterium strains each containing oneof these constructs, and transforming a plant cell with the mixedbacterial culture. An SUMT expression cassette can also be readilysubcloned into other 2 T-DNA vectors, or into a vector designed for usein microprojectile-mediated transformation, and used as anidentification sequence by a person of skill in the art.

Example 8 Use of Gene Silencing to Produce a Detectable Seed Phenotype

An inverted repeat positioned within an intron of the marker genecassette can lead to efficient gene silencing in plant cells. This isdisclosed in detail in U.S. Application Publication No. 2006/0200878(e.g., FIGS. 7, 8, 9, incorporated herein by reference). To test if adsRNA encoded by inverted repeats placed within an intron was capable ofeliciting gene silencing, inverted repeats of a ˜400 bp segment of theluciferase gene (SEQ ID NO:1) were placed into the intron of the riceActin1 promoter in a EPSPS-CP4 gene cassette (pMON73874) and the abilityof the construct to suppress the luciferase gene in a transienttransformation of corn leaf protoplasts was tested. As a control, asimilar plasmid was tested, except that the control plasmid had invertedrepeats of a segment of the GUS gene instead of the luciferase gene(pMON73875). Finally, as an additional control, pMON25492, which wasidentical except that it has no inverted repeats, was also employed(FIG. 13).

When these three plasmids were tested in a corn leaf protoplasttransient gene silencing system testing for the suppression of fireflyluciferase and normalizing to the expression of a RENILLA luciferase(Promega Corp., Madison, Wis.) internal control, it was observed thatplasmid with dsRNA encoding inverted repeats within the intron(pMON73874) was able to suppress luciferase relative to the controlspMON73875 and pMON25492 (FIG. 14). The experiment was repeated a secondtime with similar results.

To test if a corn kernel phenotype may be generated via a gene silencingapproach, constructs designed to suppress the Waxy gene were made.pMON81990 contains inverted repeats of part of the Waxy gene. Transgeniccorn plants containing pMON81990 displayed silencing of the Waxy gene inat least 65% of the independent R0 plants, as determined by stainingpollen and kernels with iodine for starch production. In comparison,plants containing pMON81993, which expresses a sense fragment of Waxy,do not display efficient silencing of the Waxy gene.

Silencing of genes that encode zeins (seed storage proteins), leading toa visible phenotype was also demonstrated. pMON73567 contains invertedrepeats of sequences of genes that encode α-zeins in corn kernels.Transcription of the inverted repeats results in silencing of thesegenes, reducing the levels of the 19 kD and 22 kD α-zeins in 26 out of29 R0 plants tested. FIG. 15 demonstrates that kernels resulting fromcells transformed with this dsRNA-encoding sequence have an obviousvisual phenotype, wherein kernels with reduced zeins are lesstranslucent than wild-type kernels.

Thus, a dsRNA-encoding sequence embedded in the intron of a marker genemay be used as an identification sequence according to the presentinvention. Constructs containing, for instance, a glyphosate resistancegene such as CP4 EPSPS as a selectable marker and such a dsRNA-encodingsequence in an intron of the selectable marker gene may be directly usedfor co-transformation with a one T-DNA construct containing a gene ofinterest by mixing cells of two Agrobacterium strains each comprisingone of these constructs and transforming a plant cell with the mixedbacterial culture. Alternatively, the dsRNA-encoding cassette may besubcloned into a 2T-DNA construct as an identification sequence, forefficient identification of marker free seeds. One of skill in the artcould also design analogous constructs for use in microprojectilebombardment-mediated plant cell transformation.

Example 9 Use of KAS4 as an Identification Sequence

Binary vector pMON83530 (FIG. 16) contains a KAS4 (a-keto-acetyl-ACTsynthase; GenBank accession AF060518) driven by a soybean USP88 promoter(e.g. U.S. Pat. No. 7,078,588) with a CP4 plant expressible cassette asa selectable marker on the same T-DNA. The seed-specific expression ofthe KAS4 gene results in shrunken seeds which are easily distinguishablefrom the normal seeds which do not contain the gene (FIG. 17). Theconstruct can be directly used as an identification sequence, and aT-DNA comprising such an identification sequence and a selectable markermay be co-transformed with a second construct comprising a T-DNAcontaining a gene of interest by mixing two Agrobacterium strains eachcontaining one of these constructs and transforming a plant cell withthe mixed bacterial culture.

The KAS4 expression cassette is also present in a 2 T-DNA plasmid asshown in pMON107314 (FIG. 18) wherein one T-DNA comprises a splA gene(Sucrose phosphorylase from Agrobacterium tumefaciens; GenBank AccessionAE009432) as an identification sequence and a marker gene and the otherT-DNA may comprise a gene of interest as constructed by routine cloningmethods known to those skilled in the art. The 2 T-DNA plasmid can thenbe used, for instance, for soybean transformation. The identificationgene is used for selecting seeds without marker gene based on phenotypeprovided by the identification gene.

Example 10 Use of an splA gene as an Identification Sequence

Binary vector pMON68581 (FIG. 19) contains the splA (Sucrosephosphorylase from Agrobacterium tumefaciens; GenBank AccessionAE009432) driven by a soybean 7S alpha promoter (e.g. GenBank M13759;Doyle et al., 1986) with a CP4 plant expressible cassette as aselectable marker on the same T-DNA. The seed-specific expression of thesplA gene results in shrunken seeds which are easily distinguishablefrom the normal seeds which do not contain the gene (FIG. 20). Theconstruct can be directly used as an identification sequence, and aT-DNA comprising such an identification sequence and a selectable markermay be co-transformed with a second construct comprising a T-DNAcontaining a gene of interest by mixing two Agrobacterium strains eachcontaining one of these constructs and transforming a plant cell withthe mixed bacterial culture. The splA expression cassette can also bereadily sub-cloned into 2 T-DNA vectors wherein one T-DNA comprises splAgene as an identification sequence and a marker gene and the other T-DNAcomprises a gene of interest by routine cloning methods known to thoseskilled in the art. The 2 T-DNA plasmid can then be used, for instance,for soybean transformation. The identification sequence is used forselecting seeds without a selectable or screenable marker gene based onthe phenotype phenotype provided by the identification sequence.

Example 11 Use of Several Identification Sequences in ProducingMarker-Free Corn Seed

Multiple 2 T-DNA plant expression vectors were constructed. In eachconstruct, the first T-DNA segment comprised a plant expressible uidAtransgene as an example of a nucleic acid of interest and the secondT-DNA segment comprised of a plant expressible CP4 EPSPS transgene as aselectable marker and an identification sequence as shown in the tablebelow. Sequences of crtB designed for expression in monocots wereprepared by methods known in the art (e.g. by codon-optimization asfound in SEQ ID NO:2; SEQ ID NO:3). pMON68412 comprises SEQ ID NO:3. Thefirst T-DNA is flanked by right and left borders while the second T-DNAis a located in the vector backbone, a 2 T-DNA format commonly known astandem format (Huang et al., 2005). Corn tissues were transformedseparately with each of the constructs by methods known in the art. Theexpected phenotype with each identification gene is indicated in Table 2below. Alternative promoters for expression of the identificationsequences in endosperm include glutelin1 promoter from rice, the waxypromoter from corn, and the brittle2 promoter from corn.

TABLE 2 Exemplary phenotypes expected with given identificationsequences. Phenotype of seeds carrying the identifica- IdentificationSequence Cassette tion gene and Identification the selectable Promotersequence Terminator Construct marker gene Maize 27 crtB Rice pMON68412carotenoid kD zein Glutelin1 pigment in endosperm; defective kerneldevelopment Maize 27 19 & 22 kD Rice pMON68413 opaque kD zein zeininverted Glutelin1 endosperm repeats (US 20060200878) Maize 27 Phospho-Rice pMON68414 shrunken kD zein fructokinase Glutelin1 endosperm (pfk)Maize 27 B peru Rice pMON68415 anthocyanin kD zein Glutelin1 pigment inendosperm None None None pMON97371 none

All of the compositions and methods disclosed and claimed herein can bemade and executed without undue experimentation in light of the presentdisclosure. While the compositions and methods of this invention havebeen described in terms of the foregoing embodiments and illustrativeexamples, it will be apparent to those of skill in the art thatvariations, changes, modifications, and alterations can be applied tothe composition, methods, and in the steps or in the sequence of stepsof the methods described herein, without departing from the concept,spirit, and scope of the invention. More specifically, it will beapparent that certain agents that are both chemically andphysiologically related may be substituted for the agents describedherein while the same or similar results would be achieved. All suchsimilar substitutes and modifications apparent to those skilled in theart are deemed to be within the spirit, scope, and concept of theinvention as defined by the appended claims.

REFERENCES

The following references, to the extent that they provide exemplaryprocedural or other details supplementary to those set forth herein, arespecifically incorporated herein by reference.

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1. A method of preparing marker-free seeds from a transgenic plantcomprising the steps of: a) obtaining seeds of a transgenic plantcomprising a first DNA segment comprising a nucleic acid of interest anda second DNA segment comprising a marker gene physically linked to a DNAcassette that is operably linked to a promoter functional in said seed,wherein the DNA cassette confers a detectable phenotype; b) screeningthe seeds for the absence of the detectable phenotype; and c) selectingat least a first seed that lacks the detectable phenotype to obtain aseed free of the marker gene.
 2. The method of claim 1, wherein step c)further comprises assaying the seed for the presence of the nucleic acidof interest and selecting a seed that comprises the nucleic acid ofinterest.
 3. The method of claim 1, wherein the marker gene is aselectable marker gene.
 4. (canceled)
 5. The method of claim 4, whereinthe DNA cassette encodes an RNA that is translationally ortranscriptionally fused to the selectable marker gene. 6-8. (canceled)9. The method of claim 1, wherein the first DNA segment and second DNAsegment overlap.
 10. The method of claim 1, wherein the seed selected instep c) lacks the marker gene and DNA cassette.
 11. The method of claim1, wherein the transgenic plant or a progenitor thereof of any previousgeneration was co-transformed with the first and second DNA segments onseparate DNA constructs.
 12. The method of claim 1, wherein the firstand second DNA segments are bounded by different T-DNA border sequences.13. The method of claim 12, wherein step a) comprises transforming thetransgenic plant or a progenitor thereof of any previous generation witha single DNA construct comprising the first and second DNA segments. 14.The method of claim 1, wherein the transgenic plant was produced bytransforming the plant or a progenitor thereof of any previousgeneration with a DNA construct comprising (i) the first DNA segmentflanked by left and right T-DNA borders comprising a nucleic acid ofinterest, and (ii) the second DNA segment flanked by a second set ofleft and right T-DNA borders, wherein the second DNA segment furthercomprises a marker gene operably linked to a promoter functional in thetransgenic plant.
 15. The method of claim 1, wherein the first andsecond DNA segments are not genetically linked in said transgenic plant.16. The method of claim 1, wherein the first and second DNA segments aregenetically linked in said transgenic plant.
 17. The method of claim 1,wherein the transgenic plant was produced by introducing the first andsecond DNA segments into said plant or a progenitor thereof of anyprevious generation by transformation mediated by a bacterial strainselected from the genus Agrobacterium, Rhizobium, Mesorhizobium, orSinorhizobium.
 18. (canceled)
 19. The method of claim 3, wherein theselectable marker gene encodes a product selected from the groupconsisting of CP4 EPSPS, phosphinothricin acetyltransferase, DMO, NptII,glyphosate acetyl transferase, mutant acetolactate synthase,methotrexate resistant DHFR, dalapon dehalogenase, PMI, Protox,hygromycin phosphotransferase and 5-methyl tryptophan resistantanthranilate synthase.
 20. The method of claim 1, wherein the DNAcassette sequence is selected from the group consisting of crtB, gus,gfp, lux, an anthocyanin synthesis gene, cobA, and B-peru.
 21. Themethod of claim 1, wherein the DNA cassette is operably linked to apromoter functional in a tissue selected from an embryo, seed endosperm,cotyledon, aleurone, and seed coat.
 22. The method of claim 1, whereinthe DNA cassette comprises a nucleic acid sequence operably linked to apromoter selected from the group consisting of a napin promoter, abeta-phaseolin promoter, a beta-conglycinin subunit promoter, a zeinpromoter, an Osgt-1 promoter, an oleosin promoter, a starch synthasepromoter, a globulin 1 promoter, a barley LTP2 promoter, analpha-amylase promoter, a chitinase promoter, a beta-glucanase promoter,a cysteine proteinase promoter, a glutaredoxin promoter, a HVA1promoter, a serine carboxypeptidase II promoter, a catalase promoter, analpha-glucosidase promoter, a beta-amylase promoter, a VP1 promoter,USP, USP88, USP99, Lectin, and a bronze2 promoter.
 23. The method ofclaim 1, wherein the detectable phenotype is selected from the groupconsisting of seed color, seed opacity, seed germinability, seedviability, seed shape and seed texture.
 24. The method of claim 1,wherein the detectable phenotype is assayed by detection of a catalyticactivity. 25-26. (canceled)
 27. The method of claim 1, wherein step c)is carried out by an automated seed sorting machine.
 28. A DNA constructcomprising (a) a first DNA segment comprising left and right T-DNAborders flanking a gene of interest operably linked to a promoterfunctional in plants, and (b) a second DNA segment comprising a secondset of left and right T-DNA borders flanking a promoter functional in aseed operably linked to a DNA cassette that confers a detectablephenotype in seeds comprising the DNA cassette and a marker geneoperably linked to a promoter functional in plants.
 29. The DNAconstruct of claim 28, wherein the marker gene is a selectable markergene.
 30. The construct of claim 28, wherein the gene of interestconfers a trait selected from the group consisting of herbicidetolerance, insect or pest resistance, disease resistance, increasedbiomass, modified fatty acid metabolism, modified carbohydratemetabolism, and modified nutritional quality.
 31. The construct of claim28, wherein the DNA cassette and selectable marker gene are operablylinked to the same promoter.
 32. The construct of claim 28, wherein theDNA cassette and the selectable marker gene are operably linked todifferent promoters.
 33. The construct of claim 28, wherein theselectable marker gene encodes a product selected from the groupconsisting of CP4 EPSPS, phosphinothricin acetyltransferase, DMO, NptII,glyphosate acetyl transferase, mutant acetolactate synthase,methotrexate resistant DHFR, dalapon dehalogenase, PMI, Protox,hygromycin phosphotransferase and 5-methyl tryptophan resistantanthranilate synthase.
 34. The construct of claim 28, wherein the DNAcassette is selected from the group consisting of crtB, gus, gfp, lux,an anthocyanin synthesis gene, cobA, and B-peru.
 35. The construct ofclaim 28, wherein the DNA cassette is operably linked to a promoterfunctional in a tissue selected from the group consisting of an embryo,seed endosperm, cotyledon, aleurone, and seed coat.
 36. The construct ofclaim 22, wherein the DNA cassette is operably linked to a promoterselected from the group consisting of a napin promoter, a beta-phaseolinpromoter, a beta-conglycinin subunit promoter, a zein promoter, anOsgt-1 promoter, an oleosin promoter, a starch synthase promoter, aglobulin 1 promoter, a barley LTP2 promoter, an alpha-amylase promoter,a chitinase promoter, a beta-glucanase promoter, a cysteine proteinasepromoter, a glutaredoxin promoter, a HVA1 promoter, a serinecarboxypeptidase II promoter, a catalase promoter, an alpha-glucosidasepromoter, a beta-amylase promoter, a VP1 promoter, a USP, USP88 or USP99promoter, and a bronze2 promoter.
 37. A transgenic cell transformed withthe construct of claim
 28. 38. A transgenic plant transformed with theconstruct of claim
 28. 39. A transgenic plant co-transformed with a DNAconstruct comprising a first DNA segment comprising left and right T-DNAborders flanking a gene of interest operably linked to a promoterfunctional in plants and a second DNA construct containing a second DNAsegment comprising a second set of left and right T-DNA borders flankinga promoter functional in a seed operably linked to a DNA cassette thatconfers a detectable phenotype in seeds comprising the DNA cassette anda marker gene operably linked to a promoter functional in plants.
 40. Acell of the plant of claim
 39. 41. A DNA construct comprising right andleft T-DNA borders, wherein a first DNA segment comprising a gene ofinterest operably linked to a promoter functional in plants is locatedafter the left border and a second DNA segment comprising a DNA cassettethat confers a detectable phenotype to plant seeds that comprise the DNAcassette and a marker gene operably linked to a promoter functional inplants is located after the right border.
 42. A DNA construct comprisingright and left T-DNA borders, wherein a first DNA segment comprising aDNA cassette that confers a detectable phenotype to plant seeds thatcomprise the DNA cassette and a marker gene operably linked to apromoter functional in plants is located after the right border, and asecond DNA segment comprising a gene of interest operably linked to apromoter functional in plants is located after the left border.
 43. ADNA construct comprising first and second right T-DNA borders, wherein afirst DNA segment comprising a gene of interest operably linked to apromoter functional in plants is located after the first right borderand a second DNA segment comprising a DNA cassette that confers adetectable phenotype to plant seeds that comprise the DNA cassette and amarker gene operably linked to a promoter functional in plants locatedafter the second right border. 44-48. (canceled)